PROJECT SUMMARY Historically, toxicity testing has relied on high dose exposures in animals with default methods for extrapolating to low level exposures in human populations, producing (at high cost) great uncertainty for human health risk assessments. This Brown University Bioengineering Research Partnership (BRP)?Human 3D Microtissues for Toxicity Testing via Integrated Imaging, Molecular and Functional Analyses? implements a transformative change by developing novel 3D human microtissues as a bridging technology based on integrating imaging, molecular and functional analyses that will facilitate more rapid, cost-effective toxicity testing of environmental chemicals and emerging toxicants. The project has two phases. Phase 1 establishes 3D microtissues of five tissue types (prostate, ovary, lung, brain, heart) to address the key challenges facing development of these predictive biology platforms: reproducibility, biological complexity, integrated endpoints, and human variability. Phase 2 selects two of these 3D microtissue models for computational systems biology analysis with sufficient dose- and time-response data to define adverse points of departure for an in vitro-to-in vivo extrapolation and safety assessment. Working with collaborators and commercial partners, the BRP team includes faculty from biology, engineering, mathematics, and medicine who have formed the Center to Advance Predictive Biology (https://www.brown.edu/research/projects/center-to-advance-predictive-biology/). The following working hypothesis guides the project: In vitro pathology assessment of human 3D microtissues within a computational systems biology framework identifies toxicant-induced adverse points of departure for safety assessment. The high content, high throughput platforms for these evaluations are 3D microtissue test systems that re-capitulate the differentiated features and characteristic cellular functions of humans tissues. Progress toward the goal of transforming toxicity testing will be made by addressing these Specific Aims: ? Specific Aim 1. Innovate the 3D microtissue platform with engineering solutions for improved well designs, confocal imaging, and high-throughput workflows ? Specific Aim 2. Optimize 3D microtissues as predictive biology platforms ? Specific Aim 3. Streamline image acquisition, reconstruction, and quantitative analysis for the in vitro pathology assessment of 3D microtissues ? Specific Aim 4. Integrate imaging, molecular, and functional endpoints within a computational systems biology framework for the purpose of human safety assessment This Brown University BRP will accelerate development and commercialization of human 3D microtissue platforms as alternatives to animal toxicity testing.